www.nature.com/mi REVIEW ARTICLE OPEN New insights into intestinal phages R. Sausset1,2, M. A. Petit1, V. Gaboriau-Routhiau1,3,4 and M. De Paepe1 The intestinal microbiota plays important roles in human health. This last decade, the viral fraction of the intestinal microbiota, composed essentially of phages that infect bacteria, received increasing attention. Numerous novel phage families have been discovered in parallel with the development of viral metagenomics. However, since the discovery of intestinal phages by d’Hérelle in 1917, our understanding of the impact of phages on gut microbiota structure remains scarce. Changes in viral community composition have been observed in several diseases. However, whether these changes reflect a direct involvement of phages in diseases etiology or simply result from modifications in bacterial composition is currently unknown. Here we present an overview of the current knowledge in intestinal phages, their identity, lifestyles, and their possible effects on the gut microbiota. We also gather the main data on phage interactions with the immune system, with a particular emphasis on recent findings. Mucosal Immunology (2020) 13:205–215; https://doi.org/10.1038/s41385-019-0250-5 INTRODUCTION has been variable and antibiotics proved to be both more efficient 1234567890();,: The human gut contains a large number of viruses, mostly and cost-effective, leading to the almost abandonment of phage bacteriophages, or phages, which infect bacteria. As other viruses, therapy in most countries (reviewed in ref. 8). With the rise of phages are classified according to their type of nucleic acid, capsid bacterial resistance to antibiotics, phage therapy has recently morphology - notably the presence or absence of a tail - and the regained interest, fueling researches on applied but also basic presence or not of an envelope. The genetic material of phages phage biology. The relatively recent discovery of the influence of consists of double-stranded (ds) or single-stranded (ss) DNA or phages in aquatic bacterial ecosystems further explains the RNA, and their genome sizes range from ∼3.5 kb (e.g., ssRNA present bloom of phage studies.9 Finally, due to increased genome of Escherichia coli phage MS2) to ∼540 kb (dsDNA awareness of the importance of the gut microbiota in human genome of Prevotella LAK phages). There is considerable diversity health, a growing number of studies are addressing the roles of among phages, but 95% of them are non-enveloped tailed dsDNA phages in the gut microbiota. Emerging views suggest that phages, or Caudovirales. Within this group, the traditional intestinal phages play important roles in health and disease by differentiation into Siphoviridae, Myoviridae, and Podoviridae shaping the co-occurring bacteriome, but also by interacting families, based on tail types, is not fully coherent with phylogeny, directly with the human immune system.10–12 Several recent and therefore progressively abandoned. In addition, new phage reviews have exhaustively reported different aspects of intestinal types are constantly discovered, and classification is currently phage biology, such as its genetic diversity,13,14 bacterial ongoing reorganization. resistance mechanisms, including CRISPR–Cas systems and other Phages are present in all microbial environments and the molecular mechanisms of phage–bacteria interactions,2,3 importance of phage predation on bacteria is evidenced by the phage–bacterium antagonistic interactions in the gastro- large repertoire of bacterial anti-phage defence mechanisms. Anti- intestinal tract (GIT),15,16 lysogeny,11 and phage interactions with phage systems include cell-surface modifications that prevent the host immune system.10,11,17 Here we aim at giving a global phage recognition (phage multiplication is highly dependent on view of current knowledge of phages in the GIT, emphasizing on the proper selection of their target bacteria, which is achieved by new results, open questions, and technical difficulties of this the recognition of a specific structure on the bacterial surface1), rapidly growing field of research. but also abortive infection mechanisms that trigger cell death upon phage infection and restriction-modification or CRISPR–Cas systems that cleave invading phage genomes (reviewed in refs. 2 COMPOSITION OF THE INTESTINAL PHAGEOME and 3). Description of intestinal phages, either from a taxonomic or The presence of phages in the intestine has been described lifestyle point of view, is still in its infancy compared with that of only 2 years after their discovery by Twort,4 when d’Hérelle5 intestinal bacteria, and encounters technical difficulties. First, viral independently discovered phages, and their therapeutic potential, genomes lack universal marker genes such as the 16SrRNA gene in the stools of patients with dysentery. Before the dawn of used for bacterial taxonomic assignment. Second, the genetic antibiotics, but also later on in the Soviet Union, phages have diversity of phages remains largely unknown, preventing been utilized to treat a variety of intestinal infections, mainly sequence-based identification of most intestinal phages. Typically, cholera6 and dysentery.7 However, the success of these treatments 75% to 99% of sequences from intestinal phages do not produce 1Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France; 2Myriade, 68 boulevard de Port Royal, 75005 Paris, France; 3Laboratory of Intestinal Immunity, INSERM UMR 1163, Institut Imagine, Paris, France and 4Université Paris Descartes-Sorbonne Paris Cité, 75006 Paris, France Correspondence: M. De Paepe ([email protected]) Received: 2 September 2019 Revised: 13 November 2019 Accepted: 5 December 2019 Published online: 6 January 2020 © The Author(s) 2020 New insights into intestinal phages R. Sausset et al. 206 Fig. 1 Phage life cycles. The production of new virions is realized either through lytic cycles for Caudovirales and Microviridae phages (left side of the figure, brown arrows) or through chronic infection in the case of filamentous phages, or Inoviridae (blue arrows). Both start with the recognition and infection of the targeted bacteria (1), followed by phage DNA replication and synthesis of new virions (2). In lytic cycles, new virions are released through bacterial lysis (3), while new virions of filamentous phages exit bacteria through a dedicated secretion apparatus, without bacterial lysis (4). Phages that reproduce only through lytic cycles are called virulent. By opposition, some phages, called temperate phages, in addition to performing either lytic or chronic cycles, are able to perform lysogenic cycles (pink arrows), whereby they enter a dormant state in the infected bacteria, the prophage state (5). The prophage, either integrated within the bacterial genome or in an episomal state, is replicated with the bacterial chromosome as long as bacteria divide (6). In some bacteria, generally when submitted to a stress, the prophage is induced and the phage resumes a lytic or a chronic cycle. significant alignments to any known viral genome.13 Finally, This phenomenon, called pseudolysogeny, is poorly described, but intestinal phages are very challenging to cultivate, notably is suspected to exist in the intestinal environment (reviewed in because their bacterial hosts are mainly strict anaerobes that are ref. 21). Temperate phages, for their part, can use two very difficult to grow. However, starting from 0.2 or 0.45 µm filtered different lifestyles, the so-called “lysis–lysogeny choice”: infection fecal samples enriched in virions, shotgun deep sequencing has is either followed by a lytic cycle, as with virulent phages, or by permitted access to the human free-phage content (which will be lysogeny, whereby the phage enters a dormant state and is called designated below virome or phageome, since it comprises mainly a prophage. In this state, the expression of most phage genes is phages). Most phages appeared to be non-enveloped DNA repressed, preventing phage multiplication, but the phage viruses, either dsDNA Caudovirales or ssDNA Microviridae.In genome is replicated passively along with the bacterial genome. addition, a recent study indicates that ssDNA filamentous phages, The prophage can be either integrated in the bacterial chromo- or Inoviridae, that reproduce through chronic infection without some or extrachromosomal, like a plasmid. Following specific cues killing their host (Fig. 1) might also constitute a significant fraction described below, prophages can be activated, leading to phage of the human gut virome.18 In contrast, RNA phages were found to lytic cycle and death of the previously lysogenic bacteria. In be rare, if not completely absent, in the intestine.19,20 consequence, when considering intestinal phages, one should In the intestine as in other environments, phages can be take into account both free-phages and prophages. distinguished on the basis of their lifestyle, independently of Using recent assembly procedures, tens to thousands of DNA taxonomy (Fig. 1). Virulent phages essentially complete lytic phage contigs can be assembled per virome sample, depending cycles, whereby each infection is followed by virion production on sequencing depth. Yet, the functional roles of phages in the and host cell lysis. Yet, in some conditions such as nutritional gut ecosystem remain difficult to apprehend, notably because stress, virulent phage multiplication can be halted for a long time. they belong to entirely new and still uncharacterized genera and Mucosal Immunology (2020) 13:205 – 215 New insights into intestinal phages R. Sausset et al. 207 ab 100 nm 1. CrAssphage 100 µm 2. Microviridae 3. Myoviridae 4. Siphoviridae c 1.00 crAss-like Microviridae 0.75 Myoviridae Other Podoviridae Siphoviridae 0.50 Unassigned Relative abundance 0.25 0.00 3 6 7 8 9 3 4 5 7 8 9 1 2 4 5 6 8 9 1 2 3 4 6 7 8 1 2 4 5 1 2 6 3 7 5 9 12 12 12 11 11 11 11 10 10 10 10 Fig. 2 Main intestinal phage types. a Epifluorescence microscopy image of a human fecal filtrate. Virus-like particles (VLPs) appear as bright dots following Sybr-gold staining of DNA (M. De Paepe, unpublished results).